DNA synthesis by ovine mammary alveolar epithelial cells: effects of heparin, epidermal growth factor related peptides and interaction with stage of pregnancy

Amphiregulin is a heparin-binding member of the epidermal growth factor (EGF) family, which we have recently shown to be expressed in sheep mammary gland. Uniquely among known EGF-like growth factors, its mitogenic activity is inhibited by soluble heparin, but heparin-like molecules on the cell surface and/or in extracellular matrix appear to be necessary for amphiregulin to exert its biological effect. In primary cultures of sheep mammary alveolar epithelial cells, heparin (1-20 mg/l) inhibited DNA synthesis in a dose-dependent manner. The extent of the inhibition was influenced by physiological state, being greater (P<0.05) in mammary cell, cultures derived from 5- to 10-week pregnant sheep (63.1 +/- 8.2%, mean +/- S.E.M., n=8) than in cultures derived from sheep which were non-pregnant (35.8 +/- 8.3% inhibition, n=6) or late, 20-week, pregnant (39.8 +/- 5.6%, n=6). Both EGF and transforming growth factor-alpha (TGF-alpha) significantly (P<0.001) increased DNA synthesis in the presence of heparin. The effect of TGF-alpha was dose-related, wholly reversing the inhibitory effect of heparin in cell cultures from non-pregnant and 20-week pregnant sheep. DNA synthesis was stimulated by amphiregulin and TGF-alpha increased the maximum response. The heparin antagonist, hexadimethrine, inhibited DNA synthesis, but, in the presence of amphiregulin, approximately equivalent concentrations of heparin overcame this inhibitory effect. In the presence of heparin, TGF-alpha showed synergistic interactions with insulin or IGF-I. The results indicate interactive effects of EGF and IGF growth factor families in sheep mammary growth.